Stereochemical studies on the hydration of monofluorofumarate and 2,3-difluorofumarate by fumarase

Abstract

Stereochemical and product analyses have been studied in our continuing work on the bioprocessing of fluorinated substrate analogues. The hydration pathways of the fumarase-catalyzed reaction on fluorofumarate lead to a product distribution of L-threo-beta-fluoromalate to oxalacetate of 1 to 16. The beta-fluoromalate product has not been previously reported. Oxalacetate formation from the initial product, alpha-fluoromalate, an alpha-fluorohydrin, proceeds by way of a direct nonenzymic decomposition path (as opposed to collapse to the enol of oxalacetate with subsequent tautomerization). Difluorofumarate is hydrated to an alpha-fluorohydrin, alpha, beta-difluoromalate, which decomposes to 3(S)-fluorooxalacetate trapped by in situ malate dehydrogenase mediated reduction to L-threo-beta-fluoromalate (2R,3S). L-threo-Fluoro[2-3H]-malate is a slow substrate for the reverse reaction as measured by labilization of 3H while the erythro isomer is barely detectable. The pathways responsible for this volatilization are discussed. Acetylenedicarboxylate hydration stereochemistry was also determined where the initial product of the reaction, the enol of oxalacetate, tautomerized and was trapped by enzymic reduction to L-malate.

The X-ray Structure of trans-3-Chloroacrylic Acid Dehalogenase Reveals a Novel Hydration Mechanism in the Tautomerase Superfamily

“…The design of this compound was based on the known ability of fumarase to hydrate acetylene dicarboxylate (35). The product, hydroxyfumarate, ketonizes to afford oxaloacetate.…”

“…The addition of water results in the unstable 3-chloro-3-hydroxypropanoate intermediate, which subsequently decomposes to yield malonate semialdehyde and HCl. The cleavage of the carbon-chlorine bond in the chlorohydrin intermediate is now a chemically favorable reaction that may not need to be further catalyzed by specific halide-binding residues (35). There is, however, a possibility that the N-terminal proline residue plays an active role in the collapse of 3-chloro-3-hydroxypropanoate by deprotonating its hydroxyl group.…”

trans -3-Chloroacrylic Acid Dehalogenase from Pseudomonas pavonaceae 170 Shares Structural and Mechanistic Similarities with 4-Oxalocrotonate Tautomerase

“…Loss of a proton from the hydroxyl at C-3 would produce the malonic acid semialdehyde, which may be facilitated by water or another proton acceptor. Hydration of monofluorofumarate by fumarase also yielded an unstable intermediate, ␣-fluorohydrin (␣-fluoromalate), which subsequently decomposes to oxaloacetate and HF (23).…”

Fluorinated Substrate Analogs: Routes of Metabolism and Selective Toxicity

“…Proof that only 3s-F-oxalacetate was generated stemmed from coupled enzymic reduction by MDH with control of the stereochemistry at C2 (55). Only 2R,3S-3-fluoromalate was detected on HPLC analysis, validating the active site orientation relative to the trans addition of H20, as shown in the equation (71). eooc-c-c-coo0…”